Using synthetic ecology to optimise methanogenic consortia for anaerobic digestion
Lead Research Organisation:
University of Edinburgh
Department Name: Sch of Biological Sciences
Abstract
Synthetic ecology is the application of rational design principles to the assembly of consortia of organisms for the purpose of performing a particular task (Pandhal and Noirel, 2014). It has special relevance to microbial biotechnology, where chemically-sophisticated biochemical transformations are often performed by a complex community of different species with complementary activities, functioning in a step-wise manner or in a close, mutually-dependent association (syntrophy). One well-developed biotechnology in which such inter-species interactions are critically important is anaerobic digestion (AD), the conversion of organic matter to biogas (methane) under anaerobic conditions by methanogenic microbial consortia (Vanwonterghem et al., 2014). AD reactors can handle diverse organic waste streams such as industrial waste, food waste and farm slurry.
Anaerobic digestion is critically reliant on the activity of methanogenic Archaea, which act in syntrophy with Bacteria carrying out secondary fermentation to produce the 1-carbon compounds and hydrogen required by the methanogens. This syntrophy provides a test system for the application of synthetic ecology approaches to the structurally-complex AD community, using cultured methanogens and secondary fermenters. Such first steps are essential if we are to understand, from the bottom-up, how the core processes in an AD system work, and ultimately to design rational synthetic AD systems. Currently only two strains of methanogens are available for lab culture; in this study, we will isolate novel methanogens and their partner secondary fermenters from a natural sediment-based system, and then apply them in a synthetic ecology approach to lab-scale methanogenic reactors.
We will improve the available pool of cultured methanogens by mining a freshwater sediment-water microcosm system developed by in our laboratory (Pagaling et al., 2014). This system contains a diverse community of methanogens, as established by 16S rRNA gene sequencing and cloning/sequencing of the mcrA gene. We will extract these novel methanogens from the microcosm communities, culture them under anaerobic conditions, and use quantitative assays to assess key kinetic parameters important in AD applications, specifically growth rate, half-saturation constant for growth on H2/CO2 or acetate and growth yield.
Secondary fermenter species will be cultured from the same microcosms on a range of primary fermentation products, and combinatorial co-culture of these with the methanogen species will then be used to test for viable syntrophic associations and their efficiency and productivity. The ability of individual species from this pool to invade other syntrophic pairs will be tested to provide an indication of the susceptibility of the associations to disruption by changes in operating conditions or invasion of external organisms in a reactor context. Finally, the best synthetic associations will be used to inoculate the communities of lab-scale reactor systems, and tested for persistence, effects on biogas production and performance stability. This approach will provide a first step towards the construction of synthetic AD communities, improving our understanding of methanogen physiology and syntrophic interactions, and will have potential for the enhancement of full-scale AD reactors by bioaugmentation.
The project will involve start-of-the-art techniques for the characterisation of complex microbial communities and novel methanogenic and fermenter species by next-generation (Illumina) DNA sequencing and fingerprinting methods. Bioinformatic and statistical pipelines will be applied for the analysis of these data. Pure culture isolation of novel methanogen species will be carried out in collaboration with the laboratory of Dr. James Chong (University of York), one of the few labs in the world with this expertise.
Anaerobic digestion is critically reliant on the activity of methanogenic Archaea, which act in syntrophy with Bacteria carrying out secondary fermentation to produce the 1-carbon compounds and hydrogen required by the methanogens. This syntrophy provides a test system for the application of synthetic ecology approaches to the structurally-complex AD community, using cultured methanogens and secondary fermenters. Such first steps are essential if we are to understand, from the bottom-up, how the core processes in an AD system work, and ultimately to design rational synthetic AD systems. Currently only two strains of methanogens are available for lab culture; in this study, we will isolate novel methanogens and their partner secondary fermenters from a natural sediment-based system, and then apply them in a synthetic ecology approach to lab-scale methanogenic reactors.
We will improve the available pool of cultured methanogens by mining a freshwater sediment-water microcosm system developed by in our laboratory (Pagaling et al., 2014). This system contains a diverse community of methanogens, as established by 16S rRNA gene sequencing and cloning/sequencing of the mcrA gene. We will extract these novel methanogens from the microcosm communities, culture them under anaerobic conditions, and use quantitative assays to assess key kinetic parameters important in AD applications, specifically growth rate, half-saturation constant for growth on H2/CO2 or acetate and growth yield.
Secondary fermenter species will be cultured from the same microcosms on a range of primary fermentation products, and combinatorial co-culture of these with the methanogen species will then be used to test for viable syntrophic associations and their efficiency and productivity. The ability of individual species from this pool to invade other syntrophic pairs will be tested to provide an indication of the susceptibility of the associations to disruption by changes in operating conditions or invasion of external organisms in a reactor context. Finally, the best synthetic associations will be used to inoculate the communities of lab-scale reactor systems, and tested for persistence, effects on biogas production and performance stability. This approach will provide a first step towards the construction of synthetic AD communities, improving our understanding of methanogen physiology and syntrophic interactions, and will have potential for the enhancement of full-scale AD reactors by bioaugmentation.
The project will involve start-of-the-art techniques for the characterisation of complex microbial communities and novel methanogenic and fermenter species by next-generation (Illumina) DNA sequencing and fingerprinting methods. Bioinformatic and statistical pipelines will be applied for the analysis of these data. Pure culture isolation of novel methanogen species will be carried out in collaboration with the laboratory of Dr. James Chong (University of York), one of the few labs in the world with this expertise.
Organisations
People |
ORCID iD |
| Andrew Free (Primary Supervisor) |
Publications
Free A
(2018)
Diversity-Function Relationships in Natural, Applied, and Engineered Microbial Ecosystems.
in Advances in applied microbiology
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/M010996/1 | 01/10/2015 | 31/03/2024 | |||
| 1720734 | Studentship | BB/M010996/1 | 01/04/2016 | 31/03/2020 |
| Description | Anaerobic digestion is a key 'green' waste to resource biotechnology that utilises various waste-streams (e.g wastewater/agricultural wastes) to generate sustainable fuel (biogas) within reactors. Through the degradation of these wastes, the microorganisms involved produce intermediate compounds which enter into metabolic pathways which produce methane rich biogas (as well as additional carbon dioxide). Natural, anaerobic environments such as water-logged soil and wetland sediments are sites where methane generating organisms ('methanogens') can convert simple compounds to methane, and may contain novel species/methanogenic communities with improved, or more resilient biogas producing potential in bioreators. During the PhD so far, techniques have been developed in order to use environmental samples from both conventional and extreme environments as methanogenic inoculum in small scale bioreactors, thereby determining the production of methane from these communities under controlled conditions. Further to this, the carbon cycling potential of these communities has been assessed and gives insights into the key role these communities play in cycling carbon in the environment. Techniques have also been developed to attempt to culture unusual organisms from these communities which may have novel biotechnological function and this goes some way to aid in overcoming challenges involved in cultivating microorganisms inhabiting extreme environments. |
| Exploitation Route | As this PhD work is part-time and it is early in the duration of the work, it is hoped that these initial results can be built upon throughout the remaining years of research. Enhancing the methane producing potential of microbial communities being studied will be key - this will be performed using similar approaches as trialed previously, but by varying reactor contents in attempts to enhance methane generation (varying reactor/environmental conditions has provided additional results in the past year, and will be taken forward for additional molecular analysis). Additionally, further DNA based analysis will be utilised to aid in overcoming challenges/limitations experienced when attempting to grow challenging environmental isolates. Lastly, the use of recently updated bioinformatics pipelines will be used to assess microbial community structure/function relationships and explore the biology/taxonomy of key methane producing species that can be used in sustainable bioenergy production, and which underpin carbon cycling in sediment environments. |
| Sectors | Energy,Environment |
| Description | School Visit (East Lothian) |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Schools |
| Results and Impact | Visited various classes at a Primary School in East Lothian as part of their whole school STEM focus in September 2018. Classes across the school had been learning about the significance of STEM subjects through active learning and whole school assemblies. I visited as part of a 'Meet a Scientist' day where I gave short presentations to ~80 pupils about my research, and additionally how microorganisms fit into the tree of life and general information about the 'world of work', particularly in the lab setting. The School reported that pupils engaged well and these talks continued their excitement in exploring STEM subject further, even after the STEM topic was finished. Again, parents noted pupils enthusiasm following the visit. Pupils were encouraged to ask questions regarding STEM (and biology/sustainability in particular). Many pupils were keen so learn about the size of microbes and the use of microscopes, as well as more specific questions regarding my research, including making sustainable products from microorganisms. Further involvement in future class topics (e.g renewable energy and biofuels and the function of DNA) have been discussed. |
| Year(s) Of Engagement Activity | 2018 |